Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation, generally through the activation of the high affinity IgE receptor (Fc-epsilon-RI). Binding of an antigen to IgE/Fc-epsilon-RI complexes initiates intracellular signaling events that lead to the release of vasoactive and inflammatory cytokines which mediate immediate and delayed allergic reactions. The signaling pathways linking Fc-epsilon-RI to human mast cell activation are, however, incompletely understood. A deeper understanding of the signaling mechanisms leading to mast cell activation by Fc-epsilon-RI and other activating receptors can give clues on treatment options for human allergic disease. Our findings in FY 2016-2017 have given us new insights into the regulation of Fc-epsilon-RI and its signaling components and into the signaling mechansisms of other receptors involved in the activation of mast cells such as KIT and ADGRE2. First, we found that human mast cells express a truncated variant of the beta subunit of the Fc-epsilon-RI (MS4A2; named t-Fc-epsilon-RI-beta) that lacks exon 3 and does not traffic to the plasma membrane, functioning in intracellular rather than in plasma transmembrane signaling. Since full length MS4A2 is important for the trafficking the Fc-epsilon-RI receptor complex to the cell surface and for amplifying Fc-epsilon-RI -induced signaling, we studied the possibility that disturbing the ratio of expression of full length MS4A2 to truncated MS4A2 could reduce responses of mast cells to Fc-epsilon-RI activation. We demonstrated that using antisense oligonucleotide (AON)-mediated exon skipping ofMS4A2exon 3 in mast cells caused a disproportionate expression of the t-Fc-epsilon-RI-beta at the expense of full-length MS4A2 resulting in reduced IgE receptors in the plasma membrane. As a consequence, mast cells were functionally unresponsive to IgE-mediated antigen challenge. Furthermore, mice treated with Vivo-Morpholino AON showed reduced mast cell mediated allergic responses. Given the recent promising results of using AONs to alter splicing in other diseases, we are hopeful that this approach can be the basis for the development of mast cell-specific treatments for allergic diseases. Second, our recent results shed new light on the regulation of human mast cell activation by identifying DJ-1, an evolutionary conserved protein reported to protect cells against oxidative damage, as an unsuspected player in Lyn activation after engagement of Fc-epsilon-RI. When the IgE/Fc-epsilon-RI complex encounters an antigen, activation of the Src kinase Lyn is the initiating step of a series of phosphorylation events which amplify signaling cascades and result ultimately in the release of mast cell mediators. However, how exactly Lyn initiates these events is not entirely clear. We demonstrated that DJ-1 translocates to lipid rafts early after activation of Fc-epsilon-RI, colocalizes with Lyn in these membrane domains and directly interacts with Lyn to facilitate its kinase activity. Our interest in DJ-1 stemmed from observations that DJ-1 is abundantly expressed in mast cell lines and that the serum levels of DJ-1 are altered in patients with atopic dermatitis and in patients with systemic mastocytosis. Although a well-described function of DJ-1 in cancer and Parkinsons disease is to protect cells from oxidative stress partly by undergoing self-oxidation thus reducing reactive oxygen species (ROS) levels, we found that DJ-1 is critical for antigeninduced degranulation, cytokine and eicosanoid production by mechanisms which appear mostly independent of its antioxidant activity and involve the direct interaction of a non-oxidized form of DJ-1 with Lyn to stabilize its full kinase activity. Further characterization of DJ-1/Lyn interaction may open new avenues of therapeutic modalities in MC-derived diseases. Third, we have demonstrated a role for sphingosine kinases in the regulation of growth of normal and neoplastic mast cells. Activation of Fc-epsilon-RI, KIT or IL-3 receptors stimulate the activity of the two isoforms of sphingosine kinase (SphK1 and SphK2) which leads to the generation of the lipid mediator sphingosine-1-phosphate (S1P). Antigen-mediated generation of S1P is important for mast cell reactivity; however, the function of KIT-generated S1P on mast cell responses is not known. Because neoplastic mast cells generally have gain of function mutations in KIT and S1P is a well-recognized mediator of growth/survival and resistance to anticancer drugs in various neoplastic cell types and cancers, we explored the role of S1P in the proliferation/survival of transformed human mast cells. Our studies suggest that targeting S1P generation in these cells may be an effective strategy to prevent growth of neoplastic mast cells with KIT mutations. This is relevant because the exploration of signaling targets other than receptor tyrosine kinase (RTK) has been a focus in the search for treatments in mastocytosis since RTK inhibitors are ineffective in patients carrying the most common activating mutation (D816V) in KIT. Lastly, we identified a mutation in the adhesion G-protein coupled receptor (aGPCR),ADGRE2, as responsible for the triggering of a physical urticaria and we are exploring the signaling mechanisms by which this receptor activates mast cell upon mechanical stimulation. Patients with vibratory urticaria (VU) experience localized hives and increased histamine levels in serum in response to a stimulus of vibratory nature. These responses to vibration are caused by hyperreactivity of skin mast cells. We found that patients with a familial form of VU harbor a missense substitution (p.C492Y) in ADGRE2 , a receptor that binds dermatan sulfate, a major component in the skin. Such mutation renders mast cells more susceptible to vibration-induced activation. However, the mechanisms of activation of ADGRE2 and how this mutation enhances the ability of mast cells to respond to a mechanical stimulus is not yet understood. Our results detailing the specific signaling pathways activated through the ADGRE2 receptor upon ligand binding and vibration will provide insights into the structural requirements for ADGE2 activation, its signaling mechanisms and possible therapeutic targets for patients with VU.